To begin, incubate the biolistic bombardment plates of yeast at 30 degrees Celsius for five to seven nights. Inoculate the tester strain of the opposite mating type in two milliliters of YPD medium overnight. The next day, transfer 150 microliters of the tester strain culture on a YPD plate.
With a sterile glass handle, spread the tester evenly on the plate's surface and let it air dry. Use velvet replicating pads and a replicator stamp to make a master plate replica of the transformed plasmid auxotrophy marker on drop-out medium plate. Then, replicate on the YPD plate containing the tester's lawn and incubate.
After 48 hours, replicate the plate containing the tester on a selective medium to detect the presence of the mitochondrial gene of interest. Compare the master plate and the corresponding colonies to identify the positive colonies. Streak the positive colonies from the master plate onto a second selective drop-out plate.
The tester lawn and the transformant cells mated and resulted in infused mitochondria. Purified positive synthetic rho-colonies were obtained from URA plates. Some positive synthetic rho-colonies lost the mitochondrial construct.
